In recent years, microdevice technologies, also referred to as microfluidics and Lab-on-a-Chip technologies, have been proposed for a number of applications in the field of bioanalytical chemistry. Microdevices hold great promise for many applications, particularly in applications that employ rare or expensive fluids, such as proteomics and genomics. The small size of the microdevices allows for the analysis of minute quantities of sample. Having the potential to integrate functions such as sample collection, sample preparation, sample introduction, separation, detection, and compound identification in one device, microdevices such as μ-total analysis systems (μ-TAS) have come to represent the main focus of academic and industrial laboratories research relating to chemical analysis tools or clinical diagnostic tools.
Microdevices having integrated components, e.g., for sample preparation, separation and detection compartments have been proposed in a number of patents. See, e.g., U.S. Pat. No. 5,500,071 to Kaltenbach et al., U.S. Pat. No. 5,571,410 to Swedberg et al., and U.S. Pat. No. 5,645,702 to Witt et al. Because such microdevices have a relatively simple construction, they are in theory inexpensive to manufacture.
Microdevices may be adapted to employ or carry out a number of different separation techniques. Capillary electrophoresis (CE), for example, separates molecules based on differences in the electrophoretic mobility of the molecules. Typically, microdevices employ a controlled application of an electric field to induce fluid flow and or to provide flow switching. In order to effect reproducible and/or high-resolution separation, a fluid sample “plug,” a predetermined volume of fluid sample, must be controllably injected into a capillary separation column or conduit. For fluid samples containing high molecular weight charged biomolecular analytes such as DNA fragments and proteins, microdevices containing a capillary electrophoresis separation conduit a few centimeters in length may be effectively used in carrying out sample separation of small volumes of fluid sample having a length on the order of micrometers. Once injected, high sensitivity detection such as laser-induced fluorescence (LIF) may be employed to resolve a separated fluorescently labeled sample component.
For samples containing analyte molecules with low electrophoretic differences, such as those containing small drug molecules, the separation technology of choice is often based on chromatography. Chromatographic separation occurs when a mobile phase carries sample molecules through a chromatography bed (stationary phase) where sample molecules interact with the stationary phase surface. The velocity at which a particular sample component travels through a chromatography bed depends on the component's partition between mobile phase and stationary phase.
There are many chromatographic techniques known in the art. For example, in reverse phase liquid chromatography, where the stationary phase offers a hydrophobic surface and the mobile phase is usually a mixture of water and organic solvent, the least hydrophobic component moves through the chromatography bed first, followed by other components, in order of increasing hydrophobicity. In other words, the chromatographic separation of sample components may be based on hydrophobicity. In isocratic liquid chromatography, the content of the mobile phase is constant throughout the separation. Gradient liquid chromatography, on the other hand, requires the content of the mobile phase to change during separation. Gradient liquid chromatography not only offers high resolution and high-speed separation of wide ranges of compounds, it also allows injection of large sample volumes without compromising separation efficiency. During the initial period when the sample is introduced, the mobile phase strength is often kept low, and the sample is trapped at the head of the liquid chromatography column bed. As a result, interfering moieties such as salts are washed away. In this regard, gradient liquid chromatography is suited to analyze fluid samples containing a low concentration of analyte moieties.
Ordinarily, capillary electrophoresis is not compatible with chromatographic techniques. However, capillary electrochromatography, a fusion of liquid chromatography and capillary electrophoresis involving the application of an electric field in order to generate electroosmotic flow, has been proposed. For example, U.S. Pat. Nos. 5,770,029 and 6,007,690 each to Nelson et al. each describes microdevices employing electroosmotic flow to drive a mobile phase through a high surface area column to achieve sample enrichment. When an electric field is applied, the electroosmotic flow moves the mobile phase through the packed column. However, the charged stationary phase surfaces, e.g., chromatographic bead surfaces, are responsible for generating electrokinetic flow and/or switching as well as separation. Accordingly, capillary electrochromatography suffers from a number of drawbacks. For example, individual control over flow switching and separation is difficult to achieve in capillary electrochromatography. In addition, it is difficult to produce appropriate surfaces for both flow switching and separation for any particular sample. Furthermore, capillary electrochromatography cannot carry out gradient chromatography with reliability, since, as the content of the mobile phase changes during separation, surface charge on the stationary phase associated with electroosmotic flow also change.
Pressure-driven flow associated with conventional liquid chromatography is useful in providing flow through packed columns. A mechanical or other type of pump is typically employed to generate pressure to drive a sample through the column. For example, when particles of 3 to 5 μm diameter are packed, a pressure drop of typically 10–30 bar/cm is used in order to maintain proper fluid flow. However, such pressure-driven flow has not been successfully employed in microdevices for separation.
Since the speed and quality of separation throughput of a microdevice is determined by the precision and accuracy of fluid flow control, there is a need for an improved microdevice that employs a introducing means to controllably introduce a predetermined volume of a fluid sample into a separation column or conduit independent from the microdevice's ability to separate the components of a fluid sample according to a component property. Sample introduction may be performed without need for an electric field. Optionally, such introduction may supplement electrokinetically driven separation. In addition, there is a need for such a microdevice wherein the introducing means represents an integrated portion of the microdevice.